The present invention relates to polyalkylene oxide-conjugated hemoglobins which substantially avoid causing hemoglobinuria in mammals. The present invention also relates to methods for separating the conjugated hemoglobins by degree of polyalkylene oxide substitution while removing endotoxins and phospholipids.
Advances have occurred in recent years in the development of hemoglobin-based blood substitutes. Such transfusional fluids serve as alternatives to whole blood or blood fractions for use as oxygen carriers and plasma expanders. The use of whole blood and blood fractions has grown increasingly disfavored because of the risk of immune or non-immune reactions and infections, such as acquired immunodeficiency syndrome.
The conjugation of polyethylene glycol (PEG) to hemoglobin reduces its antigenicity and extends its residence time in circulation. However, gross hemoglobinuria was reported by Iwashita and Ajsaka, Organ-Directed Toxic.: Chem. Indicies Mech., Proc. Symp., (Brown et al., Eds. Pergamon, Oxford, England 1981) , 97-101 in exchange-transfused rats receiving PEG-conjugates of hemoglobin monomeric subunits below 40,000 daltons. The PEG-conjugation reaction had resulted in dissociation of the hemoglobin tetramer into monomer subunits.
When conjugates having molecular weights over 50,000 daltons were infused, hemoglobinuria was not observed. However, Ajisaka and Iwashita, Biochem. Biophys. Res. Comm., 97(3), 1076-81 (1980) disclosed that these PEG-conjugates of monomeric hemoglobin subunits had P.sub.50 's between 9.5 and 12 mm Hg. Such a high oxygen affinity is inefficient for delivering oxygen to tissues.
Iwasaki and Iwashita, Artif. Organs, 10(5), 411-16 (1986) disclose the preparation of pyridoxalated PEG-hemoglobin conjugates. The conjugates have weight average molecular weights of 123,000.+-.18,000 daltons and four to five PEG conjugates per hemoglobin molecule. However, this material still exhibited a 5% excretion rate into the urine over a 24 hour period when infused into rats.
U.S. Pat. No. 4,301,144 discloses various polyalkylene oxide hemoglobin conjugates with polyalkylene oxides having molecular weights between about 300 and about 20,000 daltons. The degree of substitution is between about 4 and about 120 polyalkylene oxide conjugates per hemoglobin molecule. The conjugate is disclosed as having a circulating half-life of two to four times longer than unmodified stroma-free hemoglobin.
U.S. Pat. No. 4,412,989 discloses various effector molecule modified hemoglobins conjugated to polyalkylene oxides. The polyalkylene oxides have molecular weights between about 300 and about 20,000 daltons and a degree of substitution between about 1 and about 20 conjugates per hemoglobin. A circulating half-life of four to seven times greater than stroma-free hemoglobin is reported.
U.S. Pat. No. 4,670,417 reports the unsuitability of the hemoglobin-polyalkylene oxide conjugates of U.S. Patent No. 4,301,144 and 4,412,989 because the hemoglobin also denatures during reaction with the polyalkylene oxide. The conjugation of various hemoglobins to polyalkylene oxides with carboxyl alkylene ether groups is offered as a solution. Four to six polyalkylene oxide conjugates per hemoglobin are formed, depending upon whether a dimeric or trimeric intermolecularly crosslinked conjugate is formed. The polyalkylene oxides disclosed range in molecular weight from 300 to 20,000 daltons, and the hemoglobin can be modified with effector molecules. The conjugation of polyalkylene oxides to hemoglobin via carboxyl alkylene ether linkages, however, is commercially impractical.
Without being bound by any particular theory, it is believed that the prior art overlooked the possibility that particular low-molecular weight polyalkylene oxide-hemoglobin conjugates were a cause of hemoglobinuria. It has now been discovered that hemoglobinuria is substantially eliminated with polyalkylene oxide-hemoglobin conjugates having a molecular weight greater than 85,000 daltons and a degree of substitution of five polyalkylene oxide conjugates or greater, regardless of the linkage used between the polymer and the hemoglobin. The molecular weight feature, in combination with the degree of substitution, provides a polyalkylene-oxide conjugated hemoglobin molecule that is sterically hindered from renal filtration by shape, mass and/or charge and thus not readily causing hemoglobinuria in mammals.